Abstract

Background and Purpose—Targeting the prostaglandin I2 prostanoid (IP) receptor to reduce stroke injury has been hindered by the lack of selective drugs. MRE-269 is the active metabolite of selexipag showing a high selectivity toward the IP receptor. Selexipag has been recently approved for clinical use in pulmonary hypertension. We hypothesized that postischemic treatment with MRE-269 provides long-lasting neuroprotection with improved neurological outcomes in a clinically relevant rat stroke model.

Results—Quantitative magnetic resonance imaging data showed that postischemic MRE-269 treatment significantly reduced infarct volume compared with vehicle-treated rats at both 48 hours and 3 weeks after stroke. MRE-269 treatment resulted in a significant long-term recovery in both locomotor and somatosensory functions after middle cerebral artery occlusion, which was associated with a reduced weight loss in animals receiving the IP receptor agonist. Postischemic MRE-269 treatment reduced proinflammatory cytokines/chemokines and oxidative stress. Damage to the blood–brain barrier, as assessed by extravasation of immunoglobulin G to the ischemic brain, was significantly reduced by MRE-269, which was associated with a reduction in matrix metalloproteinase-9 activity in the brain of stroked aged rats given the IP agonist at 4.5 hours after ischemia onset.

Introduction

Prostacyclin, also known as prostaglandin I2 (PGI2), is an arachidonic acid–derived eicosanoid, which is predominantly produced by the endothelium.1 PGI2 induces vasodilation, a potent inhibitor of platelet aggregation, and reduces microvascular permeability.1 Mice deficient in PGI2 prostanoid (IP) receptor displayed exacerbated neuronal death after brain ischemia.2,3 In addition, PGI2 analogs significantly reduced ischemic brain injury.2,4–6 However, the use of PGI2 and known analogs have limited therapeutic value because of chemical instability, rapid metabolism (t½≈ 4 minutes),1 and lack of selectivity for the IP receptor resulting in unsafe side effects.7 Kuwano et al7 discovered a novel, brain-permeable, and highly selective IP agonist coded as NS-304 (selexipag), a prodrug of the active form termed MRE-269 that is most active at a low nmol/L range.8 Pharmacokinetics in rats, dogs, and humans showed a significantly improved t½ of 6 to 8 hours.7,8 After successful clinical trials,9,10 selexipag/MRE-269 has been approved for pulmonary hypertension by the Food and Drug Administration in the United States.

In an animal model of excitotoxic brain injury, MRE-269 was found to exert neuroprotective effects,11 which is a significant finding because excitotoxicity contributes to neuronal death in stroke. There is limited information about the protective mechanisms and the potential to target the IP receptor pathway to reduce stroke injury and improve long-term neurological recovery. Here, we hypothesized that postischemic treatment with MRE-269 will provide long-lasting neuroprotection with improved neurological outcomes after ischemic stroke. Using an aged rat ischemic stroke model, we found that MRE-269 significantly reduced infarct volume and dramatically improved long-term recovery of both locomotor and somatosensory functions after middle cerebral artery occlusion (MCAO). Neuroprotection of MRE-269 in ischemic stroke was through a reduction in proinflammatory cytokines/chemokines and oxidative stress. Moreover, postischemic treatment with MRE-269 significantly reduced brain matrix metalloproteinase-9 (MMP-9) activity and protected against stroke-induced blood-brain barrier (BBB) disruption. Collectively, these data suggest that targeting the IP receptor with MRE-269 is a novel strategy to reduce cerebral ischemia injury and promote long-term neurological recovery in ischemic stroke.

Methods

A detailed description of all the experiments is provided as an online-only Data Supplement. A brief description of the main methodology is provided below.

Animals and MCAO Model

All experimental procedures were in accordance with the National Institutes of Health guidelines and protocols approved by the University of Florida Institutional Animal Care and Use Committee. An a priori sample size calculation was performed using the G*Power v.3.1.3 software12 and detailed in Methods section in the online-only Data Supplement. A total of 57 aged male Sprague–Dawley rats (18–20 months, Hilltop Laboratories, Scottdale, PA) were used in this study and randomly assigned to treatment groups. Transient MCAO for 90 minutes was induced using an intraluminal silicone-coated filament method as previously described by our group.13

Experimental Design and Drug Administration

For the intravenous administration of vehicle or MRE-269, a catheter was inserted into the right femoral vein of ischemic rats at the time of MCAO surgery. MRE-269 ([4-[(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]-acetic acid; Cat. No. 10010412; Cayman Chemical, Ann Arbor, MI) was dissolved in dimethyl sulfoxide and then diluted in sterile saline. For the infarct size and neurobehavioral tests experiments, 26 rats underwent transient MCAO and were randomly assigned to vehicle or treatment group with administration of 1% dimethyl sulfoxide in saline (n=13) or MRE-269 (0.25 mg/kg, n=13) starting at 4.5 hours post-MCAO. Additional doses were given every 12 hours for the first 48 hours, and then 1 injection daily for 7 days post-MCAO. The MRE-269 dose and treatment schedule were based on our preliminary findings in young rats (3–4 months) showing that MRE-269 (0.1–0.5 mg/kg, intravenous) given at 1.5 hours after stroke onset produced a dose-dependent reduction in infarct volume as measured by 2,3,5-triphenyltetrazolium chloride staining at 48 hours (data not shown). The optimal dose of 0.25 mg/kg was, therefore, used in this study in aged rats.

For the biochemical experiments, 31 rats were randomly assigned to the following groups: sham-operation (n=5), vehicle (1% dimethyl sulfoxide, n=13), or MRE-269 (0.25 mg/kg, n=13). Drug or vehicle was given intravenously starting at 4.5 hours after stroke, and an additional dose was given at 12 hours after stroke onset. Animals were euthanized at 18 hours post-MCAO and perfused with ice-cold saline. Samples from the ipsilateral and contralateral cerebral cortices were obtained for RNA isolation, immunoblotting, and lipid peroxidation analyses.

Magnetic Resonance Imaging, Image Analysis, and Behavioral Tests

At 48 hours and 21 days after MCAO, rats treated with vehicle or MRE-269 (0.25 mg/kg) were imaged in a Bruker 4.7-T magnetic resonance imaging scanner. Behavioral tests were performed pre-MCAO and at 3, 7, 14, and 21 days post-MCAO by an independent investigator blinded to the experimental groups. Somatosensory deficits were assessed using the adhesive removal test as described previously.14 Locomotor impairments were assessed with the accelerating rotarod as described previously.15 Five rats in the vehicle and 4 rats in the MRE-269 groups died during the 21 days post-MCAO period. Further details are described in the Methods section in the online-only Data Supplement.

RNA Extraction and Real-Time Polymerase Chain Reaction

Total RNA isolation from cortical tissue, cDNA synthesis, and real-time polymerase chain reaction were performed as described previously by our group.13 Results are presented as normalized expression relative to sham-operated group. Additional details are described in the Methods section in the online-only Data Supplement.

Immunoblotting

Details of the immunoblotting technique, antibody incubations, and signal detection are provided in the Methods section in the online-only Data Supplement.

Immunoglobulin G Extravasation and MMP-9 Activity Assay

The permeability of the BBB was examined by the extravasation of IgG from the blood into the brain parenchyma using an IgG ELISA kit (Cat. No. E-25G; Immunology Consultants Laboratory, Portland, OR) according to the manufacturer’s instructions. The MMP-9 activity in cortical tissue was measured using a fluorescence resonance energy transfer peptide immunoassay as described in our previous studies.13,16

Statistical Analysis

All values were expressed as mean±SEM. Statistical analysis was performed by unpaired Student t test for comparisons between 2 groups, 1-way or 2-way ANOVA followed by Bonferroni post-tests for comparisons of multiple groups. GraphPad Prism 6 was used to conduct data analysis, and P<0.05 was considered statistically significant.

Results

Because age is the single most important risk factor in stroke,17 it is important to know whether postischemic treatment with MRE-269 is able to confer long-lasting neuroprotection in aged rats subjected to ischemic stroke. Magnetic resonance imaging–based infarct size analysis was performed at 48 hours (acute phase) and at 21 days after stroke. As shown in Figure 1, delayed treatment with MRE-269 at 0.25 mg/kg resulted in a remarkable reduction in infarct volume, which was sustained for 3 weeks after stroke.

Delayed treatment with the highly selective IP receptor agonist, MRE-269, significantly reduced infarct volume in aged rats subjected to ischemic stroke. A, Timeline of MRE-269 administration, magnetic resonance imaging (MRI) scanning and behavioral testing. B, Representative apparent diffusion coefficient (ADC), T2-weighted MRI scans and 3-dimensional (3D) reconstruction images of the ischemic lesion in vehicle- and MRE-269–treated rats are shown in B. The ischemic area was marked with a discontinuous red line in ADC and T2 images and depicted as a dark red area in the right hemisphere in 3D maps. Quantification of infarct volume at 48 h and 21 days (C) showed a marked neuroprotective effect of delayed MRE-269 administration. Data were expressed as mean±SEM, *P<0.05 vs vehicle. At 48 h, n=11 for vehicle and n=10 for MRE-269; at 21 days, n=8 for vehicle and n=9 for MRE-269, respectively. MCAO indicates middle cerebral artery occlusion.

MRE-269 treatment resulted in a significant long-term recovery in both somatosensory (Figure 2A) and locomotor functions (Figure 2B) after MCAO compared with the vehicle group, which was associated with less body weight loss (Figure 2C) in animals receiving the IP receptor agonist. To determine whether there was any association between major physiological parameters and the neuroprotective effect of MRE-269 in ischemic stroke, physiological variables including blood pH, blood oxygen (PO2), carbon dioxide saturation (PCO2), ion concentrations, blood glucose, hematocrit, hemoglobin, rectal temperature, and cerebral blood flow were measured 15 minutes before and after MCAO, respectively, as well as at 15 minutes after initial dose of vehicle (1% dimethyl sulfoxide) or MRE-269 (0.25 mg/kg) injection. As shown in the Table I and Figure I in the online-only Data Supplement, we did not observe a significant difference in these physiological parameters, including cerebral blood flow, at any time point between the vehicle- and MRE-269–treated groups. These data suggest that changes in major physiological variables are unlikely to explain the neuroprotective effects of MRE-269 in ischemic stroke.

Effect of MRE-269 on neurological function and body weight loss in aged rats subjected to transient middle cerebral artery occlusion (MCAO). The drug was given as detailed in the legend of Figure 1, and neurobehavioral analyses and body weight measurement were performed at pre- and post-MCAO. The same animals were magnetic resonance imaging scanned at 48 h and at 21 days (data presented in Figure 1). Delayed MRE-269 treatment significantly improved long-term neurological outcomes assessed by adhesive removal test (A) and rotarod (B) compared with the vehicle group from days 3 to 21 after MCAO. C, Bar graphs showing body weight loss was continuously increased from 48 h to day 21 after MCAO in the vehicle group, but MRE-269 treatment significantly attenuated body weight loss after 1 week after MCAO. Data in A through C were expressed as mean±SEM, *P<0.05, **P<0.01 and ***P<0.001 vs vehicle. Vehicle, n=8; MRE-269, n=9.

Effect of MRE-269 on Cortical Gene Expression of Cytokines and Chemokines in Aged Rats After Transient MCAO

Next we examined the effects of MRE-269 on measures of oxidative damage to the brain tissue. We first determined the protein level of gp91phox (NOX2), a glycosylated subunit of NADPH oxidase (NOX), which is a major source of superoxide generation.18 Immunoblot analyses showed a dramatic increase of the gp91phox subunit in the ipsilateral cortex of MCAO-treated aged rats at 18 hours post-MCAO compared with the sham group, and MRE-269 treatment significantly reduced the gp91phox levels induced by transient MCAO (Figure 4A and 4B). Also, we investigated the effects of delayed treatment with MRE-269 on lipid peroxidation, as assessed by quantification of 4-hydroxynonenal and 8-iso-PGF2α, 2 sensitive biomarkers of oxidative stress in ischemic brain damage.19 As shown in Figure 5A through 5D, transient MCAO resulted in dramatic increases in both 4-hydroxynonenal and 8-iso-PGF2α levels in the ischemic cerebral cortex. A 4.5-hour delayed treatment with MRE-269 significantly reduced oxidative damage induced by ischemic stroke in aged rats.

Because increased oxidative stress and proinflammatory cytokines contribute to BBB breakdown after stroke,18,20,21 we next investigated the effects of MRE-269 on ischemia-induced BBB opening. At 18 hours after ischemia, we observed a dramatic increase in IgG extravasation in the ipsilateral cerebral cortex of aged rats given the vehicle. Rats receiving MRE-269 (0.25 mg/kg; intravenous) at 4.5 hours after stroke onset had significantly less BBB damage compared with the vehicle group (Figure 6A). Because increased MMP-9 activation after stroke is a key mediator of BBB disruption,21–24 we next measured MMP-9 activity in the ipsilateral and contralateral cortices in both treatment groups. Ipsilateral MMP-9 activity was significantly increased in vehicle and MRE-269 groups when compared with either sham or their respective contralateral values (Figure 6B). Postischemic treatment with MRE-269 significantly reduced MMP-9 activity in the ischemic brain as shown in Figure 6B.

Discussion

This study demonstrated for the first time that a 4.5-hour delayed administration of MRE-269, a highly specific and clinically used IP receptor agonist, provided sustained neuroprotection and improved long-term neurological recovery in aged rats subjected to ischemic stroke. Molecular studies indicated that MRE-269 reduced proinflammatory mediators, such as IL-1β, TNF-α, and MCP-1, and reduced oxidative stress and BBB damage, which likely contributed to the observed neuroprotective effect. MRE-269 is safe in the clinical setting. Repurposing its use as a potential treatment for stroke is highly significant. The marked and long-lasting neuroprotection seen with a 4.5-hour delayed administration of MRE-269 in aged rats add a significant translational value to our findings.

This is the first study to demonstrate the sustained neuroprotective effect of an IP receptor agonist given poststroke in a clinically relevant model in aged rats. Moreover, the effects of MRE-269, a highly selective IP agonist used in the clinic, have never been studied in any animal model of brain injury. Another novel aspect of this study is our finding that a delayed administration of MRE-269 significantly reduces inflammatory mediators, oxidative stress, BBB damage, and MMP-9 activity in the postischemic brain.

Prostacyclin/IP receptor activation has potent vasodilatory effects,1 which raises the possibility that increased brain perfusion could contribute to neuroprotection by IP agonists. However, low doses of PGI2 do not alter cerebral blood flow in humans.25 Consistent with these previous reports, we did not observe significant changes in cerebral blood flow between vehicle and MRE-269 groups, which suggests that the neuroprotection by MRE-269 at a dose of 0.25 mg/kg is not because of the improvement in microcirculatory reperfusion. At low doses, MRE-269 has no effect on mean arterial blood pressure or heart rate.7

It has been reported that IP receptor gene deletion exacerbated neurological deficits and infarct size in both transient MCAO and permanent MCAO model in mice.2 Conversely, treatment with beraprost, an IP receptor agonist, significantly improved negative stroke outcomes in wild-type mice2 and reduced CA1 hippocampal neuronal death after global ischemia in aged mice.26 MRE-269 has a much higher selectivity at low nmol/L range and a longer half-life (6–8 hours) compared with the classical IP receptor agonists, such as beraprost and iloprost. More importantly, beraprost and iloprost also bind to other prostanoid receptors especially the EP3 receptor,7,8 potentially causing unsafe side effects such as marked hypotension, increased heart rate, impaired gastrointestinal function, and lung arterial contraction,7,27,28 which limits their clinical use. As a highly specific and clinically available IP receptor agonist used in the treatment of pulmonary hypertension,9,10 our data for the first time demonstrated that delayed treatment with MRE-269 reduced infarct volume in aged rats subjected to transient MCAO, suggesting a potentially valuable application in the treatment of ischemic stroke.

Because age is the single most important risk factor in stroke,17 it is of great clinical significance to know whether postischemic treatment with MRE-269 is able to confer long-lasting neuroprotection in aged rats subjected to ischemic stroke. Consistent with our findings in young rats, delayed treatment with MRE-269 (0.25 mg/kg) also exerted neuroprotective effects demonstrated by reduced infarct volume in aged rats subjected to transient MCAO. Importantly, sufficiently delayed drug administration is lacking in most experimental stroke studies which reduces their translational relevancy and may contribute to the discrepancy between the efficacy of pharmaceutical stroke treatment in animal studies and in the clinic. Also, considerable stroke research has been limited to the acute phase of focal ischemia injury in young animals rather than aged, which is less clinically relevant to most stroke populations in human. In this study, we tried to mimic a clinical setting by using aged rats tested for 3 weeks after MCAO to evaluate the efficacy of MRE-269 with an initial administration at 4.5 hours postocclusion. Delayed MRE-269 treatment resulted in a significant improvement of long-term recovery in both locomotor and somatosensory functions after MCAO. Collectively, our data provide a solid indication that postischemic intervention with MRE-269 is a promising neuroprotective therapy for ischemic stroke.

Ischemia/reperfusion triggers immune responses, excessive oxidative stress, adhesion molecule upregulation, and peripheral leukocyte recruitment, which ultimately cause inflammatory cell activation and infiltration. The activated inflammatory cells release many neuroinflammatory mediators, including cytokines, chemokines, and MMPs, thus, resulting in BBB disruption and neuronal cell death in ischemic stroke.20 In this study, we found that stroke-induced neurobehavioral deficits and infarction were associated with the upregulation of proinflammatory cytokines IL-1β, TNF-α, and IL-6 as well as MCP-1 in the ipsilateral cerebral cortex of aged rats. As reported previously in a rat distal transient MCAO model, both IL-1β and TNF-α mRNA are induced in the ischemic cortex as early as 1 hour after reperfusion, peaking between 3 and 6 hours post stroke, and remain elevated for up to 48 hours after MCAO.29 Administration of IL-1β to rats increases brain injury,30 and IL-1β–deficient mice showed smaller infarct volume compared with wild-type mice.31 Intracerebroventricular administration of recombinant TNF-α to spontaneously hypertensive rats 24 hours before 80 or 160 minutes transient MCAO exacerbated infarct size and neurological deficit, and inhibition of TNF-α with a monoclonal antibody significantly reduced brain damage after ischemic stroke.32 IL-6 is another proinflammatory cytokine that was detected as early as 3 hours after stroke onset in rats with peak concentrations after 24 hours and remained detectable for up to 14 days.33 However, IL-6–deficient mice did not show improved neurological function and infarct volume after stroke compared with wild-type mice.34 MCP-1 is one of the most commonly expressed chemokines that regulate migration and infiltration of monocytes/macrophages during neuroinflammation. Increased MCP-1 in the brain markedly exacerbated ischemic damage, which was correlated with inflammatory cell recruitment.35 Mice deficient in CCR2 (CCR2−/−), the receptor for MCP-1, had a dramatic reduction in infarct size, BBB permeability, and edema after focal transient MCAO compared with wild-type mice. The protection seen in CCR2−/− mice was associated with a marked reduction in immune cell infiltration and reduced expression/production of proinflammatory cytokines, such as IL-1β and TNF-α.36 In line with these reports, our data also found that transient MCAO significantly increased mRNA expression of IL-1β, TNF-α, MCP-1, and trends toward a considerable increase of IL-6 in the ipsilateral cerebral cortex at 18 hours post stroke. Further findings indicated that delayed treatment with MRE-269 significantly reduced the mRNA levels of IL-1β, TNF-α, and MCP-1, but not IL-6 induced by MCAO, which suggests that suppression of IL-1β, TNF-α, and MCP-1 expression by MRE-269 may be one of the mechanisms underlying the neuroprotective effects of MRE-269 in ischemic stroke.

In addition to inflammatory cytokines/chemokines, oxidative stress also plays a critical role in the pathogenesis of ischemic stroke. In the central nervous system, there are several sources generating free radicals, including the mitochondria, xanthine oxidase, uncoupled nitric oxide synthase, and cyclooxygenases.37 However, the NOX family of enzymes seems to be a major contributor to oxidative stress after ischemia. NOX2 (namely gp91phox) is a critical contributor to worse stroke outcomes because it is a major source of superoxide generation and a key contributor to ischemic injury.38,39 In this study, we found for the first time that neurological impairment and brain infarction were associated with a significant increase of gp91phox protein after stroke in the aged rat brain, and delayed MRE-269 treatment dramatically attenuated its increase. Previous studies have shown that activation of the IP receptor reduces gp91phox levels in peripheral endothelial cells.40,41 This is the first study to show reduced gp91phox levels by a PGI2 analog in stroke. In addition, a dramatic increase in 8-iso-PGF2α and 4-hydroxynonenal–modified proteins were observed in the ischemic cerebral cortex after stroke. MRE-269 significantly reduced both 8-iso-PGF2α and 4-hydroxynonenal levels in the aged ischemic brain. Collectively, these findings suggest that the neuroprotective effect of MRE-269 in stroke may be partly through the reduction of oxidative stress induced by transient MCAO.

Stroke-induced proinflammatory cytokines and free radicals have been shown to exacerbate brain injury, in part, by increasing BBB permeability.18,20 Treatment with MRE-269 resulted in less BBB opening, which was associated with a significant reduction in ischemia-induced MMP-9 activation. Although activation of the IP receptor has been shown to reduce ischemia-induced brain microvascular permeability,42,43 this is the first study showing that MRE-269 attenuates BBB damage after ischemic stroke likely by reducing MMP-9 activation.

In conclusion, we show that the delayed treatment with the highly selective IP receptor agonist, MRE-269, provided sustained protection, which correlated with a marked improvement in neurological function, reduction in infarct size, and reduced body weight loss in a stroke model in aged rats. Mechanistically, the downregulation of proinflammatory cytokines IL-1β, TNF-α, and chemokine MCP-1, as well as the reduction of oxidative damage and BBB disruption by MRE-269 likely contributed to the observed neuroprotective effect. These data strongly suggest that targeting the IP receptor with MRE-269 is a novel strategy to reduce cerebral ischemia injury and promote long-term neurological recovery in ischemic stroke. Repurposing the use of selexipag/MRE-269 for ischemic stroke is of great translational relevance because this clinically approved drug has therapeutic potential to ameliorate ischemic brain injury.

Acknowledgments

We thank Dr Kimberly E. Hawkins for her critical reading of the article and Dr Huadong Zeng for his assistance in using the magnetic resonance imaging system.

Sources of Funding

This research was supported by a Grant-in-Aid from the American Heart Association (AHA; grant number 16GRNT31020048), the NIH (R01 NS065849 to Dr Candelario-Jalil), the McKnight Brain Institute, University of Florida, and the Thomas Maren Junior Investigator Award to Dr Candelario-Jalil.